Prodrugs of CC-1065 analogs

ABSTRACT

Prodrugs of analogs of the anti-tumor antibiotic CC-1065 having a cleavable protective group such as a piperazino carbamate, a 4-piperidino-piperidino carbamate or a phosphate, in which the protecting group confers enhanced water solubility and stability upon the prodrug, and in which the prodrug also has a moiety, such as a disulfide, that can conjugate to a cell binding reagent such as an antibody. The therapeutic use of such prodrug conjugates is also described; such prodrugs of cytotoxic agents have therapeutic use because they can deliver cytotoxic prodrugs to a specific cell population for enzymatic conversion to cytoxic drugs in a targeted fashion.

FIELD OF THE INVENTION

[0001] The present invention relates to novel prodrugs of cytotoxicagents and their therapeutic uses. More specifically, the inventionrelates to novel prodrugs of cytotoxic agents that are analogs ofCC-1065 and which comprise both a moiety for chemical linkage to a cellbinding agent and a protecting group that is cleaved in vivo. Theprodrugs can be chemically linked to cell binding agents to providetherapeutic agents capable of being activated and released in vivo, anddelivered to specific cell populations in a targeted manner.

BACKGROUND OF THE INVENTION

[0002] Many reports have appeared which are directed to the targeting oftumor cells with monoclonal antibody-drug conjugates {Sela et al, inImmunoconjugates, pp. 189-216 (C. Vogel, ed. 1987); Ghose et al, inTargeted Drugs, pp. 1-22 (E. Goldberg, ed. 1983); Diener et al, inAntibody Mediated Delivery Systems, pp. 1-23 (J. Rodwell, ed. 1988);Pietersz et al, in Antibody Mediated Delivery Systems, pp. 25-53 (J.Rodwell, ed. 1988); Bumol et al, in Antibody Mediated Delivery Systems,pp. 55-79 (J. Rodwell, ed. 1988); G. A. Pietersz & K. Krauer, 2 J. DrugTargeting, 183-215 (1994); R. V. J. Chari, 31 Adv. Drug Delivery Revs.,89-104 (1998); W. A. Blattler & R. V. J. Chari, in Anticancer Agents,Frontiers in Cancer Chemotherapy, 317-338, ACS Symposium Series 796; andI. Ojima et al eds, American Chemical Society 2001}. Cytotoxic drugssuch as methotrexate, daunorubicin, doxorubicin, vincristine,vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin andmaytansinoids have been conjugated to a variety of murine monoclonalantibodies. In some cases, the drug molecules were linked to theantibody molecules through an intermediary carrier molecule such asserum albumin {Garnett et al, 46 Cancer Res. 2407-2412 (1986); Ohkawa etal, 23 Cancer Immunol. Immunother. 81-86 (1986); Endo et al, 47 CancerRes. 1076-1080 (1980)}, dextran {Hurwitz et al, 2 Appl. Biochem. 25-35(1980); Manabi et al, 34 Biochem. Pharmacol. 289-291 (1985); Dillman etal, 46 Cancer Res. 4886-4891 (1986); and Shoval et al, 85 Proc. Natl.Acad. Sci. U.S.A. 8276-8280 (1988)}, or polyglutamic acid {Tsukada etal, 73 J. Natl. Canc. Inst. 721-729 (1984); Kato et al, 27 J. Med. Chem.1602-1607 (1984); Tsukada et al, 52 Br. J. Cancer 111-116 (1985)}.

[0003] A wide array of linkers is now available for the preparation ofsuch immunoconjugates, including both cleavable and non-cleavablelinkers. In vitro cytotoxicity tests, however, have revealed thatantibody-drug conjugates rarely achieve the same cytotoxic potency asthe free unconjugated drugs. This has suggested that mechanisms by whichdrug molecules are released from conjugated antibodies are veryinefficient. Early work in the area of immunotoxins showed thatconjugates formed via disulfide bridges between monoclonal antibodiesand catalytically active protein toxins were more cytotoxic thanconjugates containing other linkers {Lambert et al, 260 J. Biol. Chem.12035-12041 (1985); Lambert et al, in Immunotoxins 175-209 (A. Frankel,ed. 1988); Ghetie et al, 48 Cancer Res. 2610-2617 (1988)}. This improvedcytotoxicity was attributed to the high intracellular concentration ofreduced glutathione contributing to the efficient cleavage of thedisulfide bond between the antibody molecule and the toxin.Maytansinoids and calicheamicin were the first examples of highlycytotoxic drugs that had been linked to monoclonal antibodies viadisulfide bonds. Antibody conjugates of these drugs have been shown topossess high potency in vitro and exceptional antitumor activity inhuman tumor xenograft models in mice {R. V. J. Chari et al., 52 CancerRes., 127-131 (1992); C. Liu et al., 93, Proc. Natl. Acad. Sci.,8618-8623 (1996); L. M. Hinman et al., 53, Cancer Res., 3536-3542(1993); and P. R. Hamann et al, 13, BioConjugate Chem., 40-46 (2002)}.

[0004] An attractive candidate for the preparation of such cytotoxicconjugates is CC-1065, which is a potent anti-tumor antibiotic isolatedfrom the culture broth of Streptomyces zelensis. CC-1065 is about1000-fold more potent in vitro than are commonly used anti-cancer drugs,such as doxorubicin, methotrexate and vincristine {B. K. Bhuyan et al.,Cancer Res., 42, 3532-3537 (1982)}.

[0005] The structure of CC-1065 (Compound 1, FIG. 1A) has beendetermined by x-ray crystallography {Martin, D. G. et al, 33 J.Antibiotics 902-903 (1980), and Chidester, C. G., et al, 103 J. Am.Chem. Soc. 7629-7635 (1981)}. The CC-1065 molecule consists of 3substituted pyrroloindole moieties linked by amide bonds. The “A”subunit has a cyclopropyl ring containing the only asymmetric carbons inthe molecule. While only the relative configuration of these carbons isavailable from x-ray data, the absolute configuration has been inferredas 3b-R, 4a-S, by using DNA as a chiral reagent {Hurley, L. H. et al,226 Science 843-844 (1984)}. The “B” and “C” subunits of CC-1065 areidentical pyrroloindole moieties.

[0006] The cytotoxic potency of CC-1065 has been correlated with itsalkylating activity and its DNA-binding or DNA-intercalating activity.These two activities reside in separate parts of the molecule. Thus, thealkylating activity is contained in the cyclopropapyrroloindole (CPI)subunit and the DNA-binding activity resides in the two pyrroloindolesubunits (FIG. 1A).

[0007] However, although CC-1065 has certain attractive features as acytotoxic agent, it has limitations in therapeutic use. Administrationof CC-1065 to mice caused a delayed hepatotoxicity leading to mortalityon day 50 after a single intravenous dose of 12.5 μg/kg {V. L. Reynoldset al., J. Antibiotics, XXIX, 319-334 (1986)}. This has spurred effortsto develop analogs that do not cause delayed toxicity, and the synthesisof simpler analogs modeled on CC-1065 has been described {M. A.Warpehoski et al., J. Med. Chem., 31, 590-603 (1988)}. In another seriesof analogs, the CPI moiety was replaced by a cyclopropabenzindole (CBI)moiety {D. L. Boger et al., J. Org. Chem., 55, 5823-5833, (1990), D. L.Boger et al., BioOrg. Med. Chem. Lett., 1, 115-120 (1991)}. Thesecompounds maintain the high in vitro potency of the parental drug,without causing delayed toxicity in mice. Like CC-1065, these compoundsare alkylating agents that bind to the minor groove of DNA in a covalentmanner to cause cell death. However, clinical evaluation of the mostpromising analogs, Adozelesin and Carzelesin, has led to disappointingresults {B. F. Foster et al., Investigational New Drugs, 13, 321-326(1996); I. Wolff et al., Clin. Cancer Res., 2,1717-1723 (1996) }. Thesedrugs display poor therapeutic effects because of their high systemictoxicity.

[0008] The therapeutic efficacy of CC-1065 analogs can be greatlyimproved by changing the in vivo distribution through targeted deliveryto the tumor site, resulting in lower toxicity to non-targeted tissues,and thus, lower systemic toxicity. In order to achieve this goal,conjugates of analogs and derivatives of CC-1065 with cell-bindingagents that specifically target tumor cells have been described {U.S.Pat. Nos. 5,475,092; 5,585,499; 5,846,545 }. These conjugates typicallydisplay high target-specific cytotoxicity in vitro, and exceptionalanti-tumor activity in human tumor xenograft models in mice {R. V. J.Chari et al., Cancer Res., 55, 4079-4084 (1995)}.

[0009] Cell-binding agents are typically only soluble in aqueous medium,and are usually stored in aqueous solutions. Thus, these analogs shouldpossess sufficient water solubility to allow for efficient reaction withcell-binding agents and subsequent formulation in aqueous solution. Inaddition, for cell-binding agent conjugates to have a useful shelf life,it is important that CC-1065 analogs that are linked to thesecell-binding agents are stable for an extended period of time in aqueoussolutions.

[0010] The CC-1065 analogs described thus far (see, e.g. FIGS. 1B and1C) are only sparingly soluble in water. Because of the sparingsolubility of CC-1065 analogs, conjugation reactions with cell-bindingagents currently have to be performed in extremely dilute aqueoussolutions. Therefore, these prodrugs should have enhanced watersolubility as compared to the parent drugs.

[0011] Also, CC-1065 analogs that have been described thus far are quiteunstable in aqueous solutions for the following reason. The seco-form ofthe drug is spontaneously converted into the cyclopropyl form, whichthen may alkylate DNA, if present. However, the competing reaction ofthe cyclopropyl form with water results in opening of the cyclopropylring to yield the hydroxy compound, which is inactive. Thus, there is aneed to protect the reactive portion of CC-1065 analogs in order toextend their useful life in aqueous solution, for example by thedevelopment of prodrugs of CC-1065 analogs.

[0012] There is therefore a need to develop prodrugs of CC-1065 analogsthat are very stable upon storage in aqueous solutions. Preferably,these prodrugs should only be converted into active drugs in vivo. Oncethe prodrug is infused into a patient, it should preferably beefficiently converted into active drug.

[0013] Carzelesin is a prodrug where the phenolic group in adozelesin isprotected as a phenyl carbamate {L. H. Li et al., Cancer Res., 52,4904-4913 (1992)}. However, this prodrug is too labile for therapeuticuse, and also affords no increase in water solubility compared to theparental drug. In a second example, the phenolic residue of a CC-1065analog was glycosylated to produce a prodrug (U.S. Pat. No. 5,646,298).However, this prodrug is not converted into active drug in vivo, andrequires the additional administration of an enzyme from a bacterialsource to convert it to the cytotoxic form.

[0014] There are a few examples of anticancer drugs, unrelated toCC-1065, that have been converted into water soluble prodrugs. In theanticancer drug irinotecan, the phenolic group is protected by a4-piperidino-piperidino carbamate. It has been reported that thisprotecting group confers water solubility to the drug. In addition, theprodrug is readily converted in vivo in humans to the active drug,presumably by the enzyme carboxylesterase, which naturally exists inhuman serum, tumor tissue and in some organs {A. Sparreboom, 4, Clin.Cancer Res., 2747-2754 (1998). L. P. Rivory et al., 52, BiochemPharmacol., 1103-1111 (1996)}.

[0015] Similarly, the anticancer drug etoposide phosphate is an exampleof a prodrug that has a phosphate protecting group and is rapidlyconverted into active drug in vivo, presumably through hydrolysis byendogenous alkaline phosphatase {S. Z. Fields et al., 1 Clin. CancerRes., 105-111 (1995)}.

[0016] Thus, there exists a need for analogs of CC-1065 that haveincreased solubility and stability in aqueous solution, to facilitatetheir conjugation to cell binding agents in aqueous solutions, whilepreserving their biological activity. In addition, in order to reducetoxic side-effects, it would be advantageous to provide the CC-1065analog in the form of a prodrug that is converted to the cytotoxic drugpredominantly at the desired therapeutic site and preferably through theaction of endogenous agents. All these advantages and more are providedby the invention described herein, as will be apparent to one of skillin the art upon reading the following disclosure and examples.

SUMMARY OF THE INVENTION

[0017] The object of the present invention is to provide prodrugs ofCC-1065 analogs, which have enhanced stability and solubility in aqueousmedium. This and other objects have been achieved by providing prodrugsin which the phenolic group of the alkylating portion of the molecule isprotected with a functionality that renders the drug stable upon storagein aqueous solution. In addition, the protecting group confers increasedwater solubility to the drug compared to an unprotected analog. Theprotecting group is readily cleaved in vivo to give the correspondingactive drug. In the prodrugs described herein, the phenolic substituentis preferentially protected as a piperazino carbamate, a4-piperidino-piperidino carbamate or as a phosphate, each of whichpossesses a charge at physiological pH, and thus have enhanced watersolubility. In order to further enhance water solubility, an optionalpolyethylene glycol spacer has been introduced into the linking groupbetween the terminal indolyl subunit C and the cleavable linkage such asa disulfide group. The introduction of this spacer does not alter thepotency of the drug.

[0018] A more specific embodiment of the invention provides a prodrugthat comprises an analog of a seco-cyclopropabenzindole-containingcytotoxic drug that has a protecting group, which enhances watersolubility and stability and that can be cleaved in vivo. The prodrug ofthis specific embodiment has a first and a second subunit that arelinked by an amide bond from the secondary amino group of the pyrrolemoiety of the first subunit to the C-2 carboxyl of the second subunit.The first subunit is shown as formula (I), and is conjugated to thesecond subunit, which is selected from among formulae (II)-(IX):

[0019] in which R represents a linking group that provides for linkageof the prodrug to a cell binding agent, where such linkage is preferablyvia a disulfide bond. The linking group may comprise a polyethyleneglycol spacer. R₁-R₆ are each independently hydrogen, C₁-C₃ linearalkyl, methoxy, hydroxyl, primary amino, secondary amino, tertiaryamino, or amido. R₇ is the protecting group that can be cleaved in vivoand enhances water solubility of the cyclopropabenzindole-containingcytotoxic drug, and is preferably a piperazino carbamate, a4-piperidino-piperidino carbamate or a phosphate.

[0020] The prodrugs of the invention can be used in cytotoxic conjugatesin which a cell binding agent is linked to one or more of the prodrugsof the present invention. Cell binding agents include antibodies andfragments thereof, interferons, lymphokines, vitamins, hormones andgrowth factors. Pharmaceutical compositions containing such conjugatesare also provided.

[0021] The cytotoxic conjugates can be used in a method for treating asubject by administering an effective amount of the above pharmaceuticalcomposition. According to the cell-type to which the selected cellbinding agent binds, many diseases may be treated either in vivo, exvivo or in vitro. Such diseases include, for example, the treatment ofmany kinds of cancers, including lymphomas, leukemias, cancer of thelung, breast, colon, prostate, kidney, pancreas, and the like.

[0022] Thus, there are provided prodrugs of CC-1065 analogs that haveimproved solubility and stability in aqueous solution, and which retaincytotoxicity when activated to produce an alkylating drug, and which areuseful in the targeting of specific cell types by means of conjugationto a specific cell binding agent.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1A shows the structure of CC-1065 and its subunits A, B, andC.

[0024]FIG. 1B and FIG. 1C show the structures of two known analogs ofCC-1065.

[0025]FIG. 2 shows the structures of exemplary CC-1065 analogs andprodrugs of the present invention.

[0026]FIG. 3 shows the structures of exemplary polyethyleneglycol-containing prodrugs of the present invention.

[0027]FIG. 4 A and B are synthesis schemes for preparing(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-methylpiperazino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC2) and(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-piperidino-piperidino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC3).

[0028]FIG. 5 shows schemes for the synthesis of PEGylated versions ofDC1, DC2 and DC3, which are DC5, DC6 and DC7, respectively.

[0029]FIG. 6 shows two synthetic schemes for the preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(phosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC4)).

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present inventors have found that the stability, watersolubility and utility of certain CC-1065 analogs are enhanced byprotection of the alkylating moiety of the analog with a suitableprotecting group. The inventors have thereby provided prodrugs ofCC-1065 analogs having enhanced aqueous solubility and stability andwhich are further capable of linkage to cell binding agents whereby thetherapeutic efficacy of such prodrugs of CC-1065 analogs is improved bychanging the in vivo distribution through targeted delivery of theprodrug to the tumor site, resulting in a lower toxicity to non-targetedtissues, and hence lower systemic toxicity. Upon delivery of theprodrug, endogenous substances substantially convert the prodrug to itsactive drug form, and, in embodiments having a cleavable linker to thecell binding agent, the active drug form of the CC-1065 analog isreleased, thus further enhancing its cytotoxic activity. Alternatively,the linker to the cell binding agent may be first cleaved inside thetarget cell to release the prodrug, followed by endogenous conversioninto the active drug.

[0031] In order to achieve this goal, the inventors synthesizedexemplary prodrugs (FIGS. 2-6) of CC-1065 analogs that areseco-cyclopropabenzindole (CBI)-containing cytotoxic prodrugscomprising: (a) a first subunit of formula (I) that is protected at thephenolic hydroxyl by a protecting group to enhance stability and watersolubility and which is cleaved in vivo, and (b) a second subunit havingthe structure represented by one of formulae (II)-(IX) and whichcomprises a linking group for conjugation of the prodrug to a cellbinding agent. The linking group can contain a polyethylene glycolspacer (FIG. 3). Removal of the protecting group of the prodrug producesan active form of the drug that retains the high cytotoxicity of theparent drug. The linker is used for conjugation to cell binding agents,preferably via a disulfide bond.

[0032] It has previously been shown that the linkage of highly cytotoxicdrugs to antibodies using a cleavable link, such as a disulfide bond,ensures the release of fully active drug inside the cell, and that suchconjugates are cytotoxic in an antigen specific manner {R. V. J. Chariet al, 52 Cancer Res. 127-131 (1992); R. V. J. Chari et al., 55 CancerRes. 4079-4084 (1995); and U.S. Pat. Nos. 5,208,020 and 5,475,092}. Inthe present invention, the inventors describe the synthesis of prodrugsof CC-1065 analogs, procedures for their conjugation to monoclonalantibodies and for measurement of the in vitro cytotoxicity andspecificity of such conjugates. Thus the invention provides usefulcompounds for the preparation of therapeutic agents directed to theelimination of diseased or abnormal cells that are to be killed or lysedsuch as tumor cells, virus infected cells, microorganism infected cells,parasite infected cells, autoimmune cells (cells that produceauto-antibodies), activated cells (those involved in graft rejection orgraft vs. host disease), or any other type of diseased or abnormalcells, while exhibiting minimal side effects.

[0033] Thus, this invention teaches the synthesis of prodrug analogs andderivatives of CC-1065 that can be chemically linked to a cell bindingagent and that maintain, upon release of the protective group, the highcytotoxicity of the parent compound CC-1065. Further, upon activation,these compounds when linked to a cell binding agent are cytotoxic tocells to which the cell binding agent binds and are much less toxic tonon-target cells.

[0034] Prodrugs of the Present Invention

[0035] The prodrugs according to the present invention comprise ananalog of CC-1065 in which the phenolic group of the alkylating portionof the molecule is protected and the prodrug further comprises a linkercapable of conjugating the prodrug to a cell binding agent. The prodrugmay comprise a first and a second subunit that are linked via an amidebond.

[0036] According to certain embodiments of the present invention, theprodrug of the CC-1065 analog has a first subunit that is a seco-CBI(cyclopropabenzindole unit) in its open chloromethyl form, wherein thefirst subunit has a phenolic hydroxyl that is protected by awater-soluble protecting group that can be cleaved in vivo. The secondsubunit of the prodrug of certain embodiments of the present inventioncomprises an analog of the combined B and C subunits of CC-1065 (FIG. 1)that are 2-carboxy-indole or 2-carboxy-benzofuran derivatives, or both,and are represented by formulae (II)-(IX). As may be ascertained fromthe natural CC-1065 and from the properties of the analogs that havebeen published {e.g. Warpehoski et al, 31 J. Med. Chem. 590-603 (1988),Boger et al, 66 J. Org. Chem. 6654-6661 (2001)}, the B and C subunitscan also carry different substituents at different positions on theindole or benzofuran rings, corresponding to positions R₁-R₆ of formulae(II)-(IX), and still retain potent cytotoxic activity.

[0037] In order to link the prodrug of the CC-1065 analog to acell-binding agent, the prodrug must first include a moiety that allowsthe derivatives to be linked to a cell binding agent via a cleavablelinkage such as a disulfide bond, an acid-labile group, a photo-labilegroup, a peptidase-labile group, or an esterase-labile group. Theprodrug analogs are prepared so that they contain a moiety necessary tolink the analog to a cell binding agent via, for example, a disulfidebond, an acid-labile group, a photo-labile group, a peptidase-labilegroup, or an esterase-labile group. In order to further enhancesolubility in aqueous solutions, the linking group can contain apolyethylene glycol spacer (FIG. 3).

[0038] Preferably, a disulfide linkage is used because the reducingenvironment of the targeted cell results in cleavage of the disulfideand release of the prodrug (or drug, depending on the relative sequenceof cleavage of the prodrug from the cell binding agent and hydrolysis ofthe protecting group), with an associated increase in cytotoxicity.

[0039] More specifically, according to certain embodiments of thepresent invention, the prodrug of an analog of CC-1065 comprises firstand second subunits that are covalently linked via an amide bond fromthe secondary amino group of the pyrrole moiety of the first subunit tothe C-2 carboxy group of the second subunit having the formulae(II)-(IX).

[0040] Within formulae (II)-(IX), R represents a moiety that enableslinkage of the prodrug of a CC-1065 analog to a cell binding agent. Thelinking moiety may contain a polyethylene glycol spacer. Examplesinclude moieties that enable linkages via disulfide bond, an acid-labilegroup, a photo-labile group, a peptidase-labile group, or anesterase-labile group, and are well-known in the art {see, e.g., U.S.Pat. No. 5,846,545, which is incorporated herein by reference}.Preferred moieties are those that enable linkage via a disulfide bond,for example a thiol (DC1, DC2, DC3, DC4, DC5, DC6, DC7) or a disulfide(DC1-SMe, DC2-SMe, DC3-SMe, DC4-SMe, DC5-SMe, DC6-SMe, DC7-Sme, seeFIGS. 2-6). Mixed disulfides containing any terminal leaving group, suchas thiomethyl (DC1-SMe, DC2-SMe, DC3-SMe, DC4-SMe, DC5-SMe, DC6-SMe,DC7-SMe), glutathione, alkyl thiol, thiopyridyl, aryl thiol, and thelike may be used provided that such disulfides are capable of undergoinga disulfide-exchange reaction for the coupling of the prodrug to a cellbinding agent. R can optionally further comprise a spacer regioninterposed between the reactive group of the linkage-enabling portionand the 2-carboxy-indole or 2-carboxy-benzofuran derivative portion.Preferred embodiments include NHCO(CH₂)_(m)SZ, NHCOC₆H₄ (CH₂)_(m)SZ, orO(CH₂)_(m)SZ, NHCO(CH₂)_(m)(OCH₂CH₂)_(n)SZ, NHCOC₆H₄(CH₂)_(m)(OCH₂CH₂)_(n)SZ, or O(CH₂)_(m)(OCH₂CH₂)_(n)SZ wherein: Zrepresents H or SR₈, wherein R₈ represents methyl, linear alkyl,branched alkyl, cyclic alkyl, simple or substituted aryl orheterocyclic, and m represents an integer of 1 to 10, n represents aninteger of 4 to 1000. Examples of linear alkyls represented by R₈include methyl, ethyl, propyl, butyl, pentyl and hexyl. Examples ofbranched alkyls represented by R₈ include isopropyl, isobutyl,sec.-butyl, tert.-butyl, isopentyl and 1-ethyl-propyl. Examples ofcyclic alkyls represented by R₈ include cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl. Examples of simple aryls represented by R₈include phenyl and naphthyl. Examples of substituted aryls representedby R₈ include aryls such as phenyl or naphthyl substituted with alkylgroups, with halogens, such as Cl, Br, F, nitro groups, amino groups,sulfonic acid groups, carboxylic acid groups, hydroxy groups and alkoxygroups. Heterocyclics represented by R₈ are compounds wherein theheteroatoms are selected from O, N, and S, and examples include furyl,pyrrollyl, pyridyl, (e.g., a 2-substituted pyrimidine group) andthiophene. Most preferred embodiments of R include NHCO(CH₂)₂SH andNHCO(CH₂)₂SSCH₃.NHCO(CH₂)₂(OCH₂CH₂)_(n)SH andNHCO(CH₂)₂(OCH₂CH₂)_(n)SSCH₃.

[0041] Within formulae (II)-(IX), R₁ to R₆, which may be the same ordifferent, independently represent hydrogen, C₁-C₃ linear alkyl,methoxy, hydroxyl, primary amino, secondary amino, tertiary amino, oramido. Examples of primary amino group-containing substituents aremethyl amino, ethyl amino, and isopropyl amino. Examples of secondaryamino group-containing substituents are dimethyl amino, diethyl amino,and ethyl-propyl amino. Examples of tertiary amino group-containingsubstituents are trimethyl amino, triethyl amino, andethyl-isopropyl-methyl amino. Examples of amido groups includeN-methyl-acetamido, N-methyl-propionamido, N-acetamido, andN-propionamido.

[0042] Within formulae (II)-(IX), R₇ is an in vivo-cleavable protectinggroup that enhances water solubility of theseco-cyclopropabenzindole-containing cytotoxic drug. Examples ofpreferred in vivo-cleavable protecting groups are piperazino carbamate,a 4-piperidino-piperidino carbamate and a phosphate, and derivativesthereof. Thus, piperazino carbamate and 4-piperidino-piperidinocarbamate protecting groups are enzyme-cleavable by enzymes such ascarboxyl esterase, which occurs in serum and plasma. Phosphateprotecting groups are cleavable by phosphatase enzymes such as alkalinephosphatase.

[0043] Disulfide-containing and mercapto-containing prodrugs of CC-1065analogs of the present invention can be evaluated for their ability tosuppress proliferation of various unwanted cell lines in vitro onlyafter they have been activated. For example, phosphoryl group-containingprodrugs, such as DC4, can be activated by incubation with commerciallyavailable alkaline phosphatases, while carbamate-containing prodrugs,such as DC3 and DC4, can be activated by incubation with commerciallyavailable carboxyl esterases. Cell lines such as, for example, the humanepidermoid carcinoma line KB, the human breast tumor line SK-BR-3, andthe Burkitt's lymphoma line Namalwa can easily be used for theassessment of the cytotoxicity of these compounds. Cells to be evaluatedcan be exposed to the compounds for 24 hours and the surviving fractionsof cells measured in direct assays by known methods. IC₅₀ values canthen be calculated from the results of the assays.

[0044] Preparation of Cell Binding Agents

[0045] The effectiveness of the prodrug compounds of the invention astherapeutic agents depends upon the careful selection of an appropriatecell binding agent. Cell binding agents may be of any kind presentlyknown, or that become known, and include peptides and non-peptides.Generally, these can be antibodies (especially monoclonal antibodies) ora fragment of an antibody that contains at least one binding site,lymphokines, hormones, growth factors, nutrient-transport molecules(such as transferrin), or any other cell binding molecule or substance.More specific examples of cell binding agents that can be used include:

[0046] monoclonal antibodies;

[0047] single chain antibodies;

[0048] fragments of antibodies such as Fab, Fab′, F(ab′)₂ andF_(v){Parham, 131 J. Immunol. 2895-2902 (1983); Spring et al, 113 J.Immunol. 470-478 (1974); Nisonoff et al, 89 Arch. Biochem. Biophys.230-244 (1960)};

[0049] interferons;

[0050] peptides;

[0051] lymphokines such as IL-2, IL-3, IL-4, L-6;

[0052] hormones such as insulin, TRH (thyrotropin releasing hormones),MSH (melanocyte-stimulating hormone), steroid hormones, such asandrogens and estrogens;

[0053] growth factors and colony-stimulating factors such as EGF, TGFα,insulin like growth factor (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF{Burgess, 5 Immunology Today 155-158 (1984)}; vitamins, such as folateand

[0054] transferrin {O'Keefe et al, 260 J. Biol. Chem. 932-937 (1985)}.

[0055] Monoclonal antibody technology permits the production ofextremely selective cell binding agents in the form of specificmonoclonal antibodies. Particularly well known in the art are techniquesfor creating monoclonal antibodies produced by immunizing mice, rats,hamsters or any other mammal with the antigen of interest such as theintact target cell, antigens isolated from the target cell, whole virus,attenuated whole virus, and viral proteins such as viral coat proteins.

[0056] Selection of the appropriate cell binding agent is a matter ofchoice that depends upon the particular cell population that is to betargeted, but in general monoclonal antibodies are preferred if anappropriate one is available.

[0057] For example, the monoclonal antibody MY9 is a murine IgG₁antibody that binds specifically to the CD33 Antigen {J. D. Griffin etal 8 Leukemia Res., 521 (1984)} and can be used if the target cellsexpress CD33 as in the disease of acute myelogenous leukemia (AML).Similarly, the monoclonal antibody anti-B4 is a murine IgG₁, that bindsto the CD19 antigen on B cells {Nadler et al, 131 J. Immunol. 244-250(1983)} and can be used if the target cells are B cells or diseasedcells that express this antigen such as in non-Hodgkin's lymphoma orchronic lymphoblastic leukemia.

[0058] Additionally, GM-CSF which binds to myeloid cells can be used asa cell binding agent to diseased cells from acute myelogenous leukemia.IL-2, which binds to activated T-cells, can be used for prevention oftransplant graft rejection, for therapy and prevention ofgraft-versus-host host disease, and for the treatment of acute T-cellleukemia. MSH, which binds to melanocytes, can be used for the treatmentof melanoma.

[0059] Preparation of Prodrug Conjugates

[0060] Conjugates of the prodrugs and a cell binding agent can be formedusing any techniques presently known or later developed. An indolyl,benzofuranyl, bis-indolyl, bis-benzofuranyl, indolyl-benzofuranyl, orbenzofuranyl-indolyl derivative coupled to the seco-CBI analog can beprepared to contain a free amino group and then linked to an antibody orother cell binding agent via an acid labile linker, or by a photolabilelinker. The prodrug compounds can be condensed with a peptide having asuitable sequence and subsequently linked to a cell binding agent toproduce a peptidase labile linker. Cytotoxic compounds can be preparedto contain a primary hydroxyl group, which can be succinylated andlinked to a cell binding agent to produce a conjugate that can becleaved by intracellular esterases to liberate free prodrug. Preferably,the prodrug compounds are synthesized to contain a free or protectedthiol group, with or without a PEG-containing spacer, and then one ormore disulfide or thiol-containing prodrugs are each covalently linkedto the cell binding agent via a disulfide bond.

[0061] Representative conjugates of the invention are conjugates ofprodrugs of CC-1065 analogs with antibodies, antibody fragments,epidermal growth factor (EGF), melanocyte stimulating hormone (MSH),thyroid stimulating hormone (TSH), estrogen, estrogen analogs, androgen,and androgen analogs.

[0062] Representative examples of the preparation of various conjugatesof prodrugs of CC-1065 analogs and cell binding agents are describedbelow.

[0063] Disulfide Linkers:

[0064] Antibody N901 which binds to the CD-56 antigen that is expressedon the surface of small cell lung cancer cells {J. D. Griffin, T.Hercend, R. Beveridge & S. F. Schlossman, J. Immunol, 130:2947 (1983)}can be used for the preparation of conjugates. The antibody is modifiedwith N-succinimidyl-3-pyridyldithio propionate as previously described{J. Carlsson, H. Drevin & R. Axen, Biochem. J., 173:723 (1978)} tointroduce, on the average, 4 pyridyldithio groups per antibody molecule.The modified antibody is reacted with the thiol-containing prodrug toproduce a disulfide-linked conjugate.

[0065] Acid-Labile Linkers:

[0066] Amino group-containing prodrugs of the present invention can belinked to antibodies and other cell binding agents via an acid labilelinker as previously described. {W. A. Blattler et al, Biochemistry 24,1517-1524 (1985); U.S. Pat. Nos. 4,542,225, 4,569,789, 4,618,492,4,764,368}.

[0067] Similarly, an hydrazido group-containing prodrug of the presentinvention can be linked to the carbohydrate portion of antibodies andother cell binding agents via an acid labile hydrazone linker {forexamples of hydrazone linkers see B. C. Laguzza et al, J. Med. Chem.,32, 548-555 (1989); R. S. Greenfield et al, Cancer Res., 50, 6600-6607(1990)}.

[0068] Photo-Labile Linkers:

[0069] Amine group containing prodrugs of the present invention may belinked to antibodies and other cell binding agents via a photolabilelinker as previously described {P. Senter et al, Photochemistry andPhotobiology, 42, 231-237 (1985); U.S. Pat. No. 4,625,014}.

[0070] Peptidase-Labile Linkers:

[0071] Amine group containing prodrugs of the present invention may alsobe linked to cell binding agents via peptide spacers. It has beenpreviously shown that short peptide spacers between drugs andmacromolecular protein carriers are stable in serum but are readilyhydrolyzed by intracellular peptidases {A. Trouet et al, Proc. Natl.Acad. Sci., 79, 626-629 (1982)}. The amino group containing containingprodrugs may be condensed with peptides using condensing agents such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDC-HCl) to give apeptide derivative that can be linked to cell binding agents.

[0072] Esterase-Labile Linkers:

[0073] Prodrugs of the present invention bearing a hydroxy alkyl groupmay be succinylated with succinic anhydride and then linked to a cellbinding agent to produce a conjugate that can be cleaved byintracellular esterases to liberate free drug. {For examples see E.Aboud-Pirak et al, Biochem Pharmacol., 38, 641-648 (1989)}.

[0074] The conjugates made by the above methods can be purified bystandard column chromatography or by HPLC.

[0075] Preferably conjugates between monoclonal antibodies or cellbinding agents and prodrugs of the present invention are those that arejoined via a disulfide bond, as discussed above. Such cell bindingconjugates are prepared by known methods such as modifying monoclonalantibodies with succinimidyl pyridyl-dithiopropionate (SPDP) {Carlssonet al, 173 Biochem. J. 723-737 (1978)}. The resulting thiopyridyl groupis then displaced by treatment with thiol containing prodrug to producedisulfide linked conjugates. Conjugates containing 1 to 10 prodrugslinked via a disulfide bridge are readily prepared by this method.Conjugation by this method is fully described in U.S. Pat. No.5,585,499, which is incorporated by reference.

[0076] In Vitro Cytotoxicity of Conjugates Between Cell Binding Agentsand Prodrugs of the Present Invention

[0077] Cytotoxicity of the prodrugs of the present invention and theirconjugates with cell binding agents can be measured after cleavage ofthe protecting group and conversion into the active drug. Cytotoxicityto non-adherent cell lines such as Namalwa and SW2 can be measured byback-extrapolation of cell proliferation curves as described inGoldmacher et al, 135 J. Immunol. 3648-3651 (1985). Cytotoxicity ofthese compounds to adherent cell lines such as A-375 and SCaBER can bedetermined by clonogenic assays as described in Goldmacher et al, 102 J.Cell Biol. 1312-1319 (1986).

[0078] Therapeutic Agent and Method for Inhibiting the Growth ofSelected Cell Populations

[0079] The present invention also provides a therapeutic agent forinhibiting the growth of selected cell populations comprising:

[0080] (a) a cytotoxic amount of one or more of the above-describedprodrugs linked to a cell binding agent, and

[0081] (b) a pharmaceutically acceptable carrier, diluent or excipient.

[0082] Similarly, the present invention provides a method for inhibitingthe growth of selected cell populations comprising contacting a cellpopulation or tissue suspected of containing cells from said selectedcell population with a cytotoxic amount of a cytotoxic agent comprisingone or more of the above-described prodrugs linked to a cell bindingagent.

[0083] The cytotoxic agent is prepared as described above.

[0084] Suitable pharmaceutically acceptable carriers, diluents, andexcipients are well known and can be determined by those of skill in theart as the clinical situation warrants.

[0085] Examples of suitable carriers, diluents and/or excipientsinclude: (1) Dulbecco's phosphate buffered saline, pH about 7.4,containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose.

[0086] The method for inhibiting the growth of selected cell populationscan be practiced in vitro, in vivo, or ex vivo.

[0087] Examples of in vitro uses include treatments of cell cultures inorder to kill all cells except for desired variants that do not expressthe target antigen; or to kill variants that express undesired antigen.

[0088] The conditions of non-clinical in vitro use are readilydetermined by the skilled artisan.

[0089] Examples of ex vivo uses include treatments of autologous bonemarrow prior to their transplant into the same patient in order to killdiseased or malignant cells: treatments of bone marrow prior to theirtransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD).

[0090] Clinical ex vivo treatment to remove tumor cells or lymphoidcells from bone marrow prior to autologous transplantation in cancertreatment or in treatment of autoimmune disease, or to remove T cellsand other lymphoid cells from allogeneic bone marrow or tissue prior totransplant in order to prevent GVHD, can be carried out as follows. Bonemarrow is harvested from the patient or other individual and thenincubated in medium containing serum to which is added the cytotoxicagent of the invention, concentrations range from about 10 μM to 1 pM,for about 30 minutes to about 48 hours at about 37° C. The exactconditions of concentration and time of incubation (=dose) are readilydetermined by the skilled artisan. After incubation the bone marrowcells are washed with medium containing serum and returned to thepatient by i.v. infusion according to known methods. In circumstanceswhere the patient receives other treatment such as a course of ablativechemotherapy or total-body irradiation between the time of harvest ofthe marrow and reinfusion of the treated cells, the treated marrow cellsare stored frozen in liquid nitrogen using standard medical equipment.

[0091] For clinical in vivo use, the cytotoxic agent of the inventionwill be supplied as solutions that are tested for sterility and forendotoxin levels or as a lyophilized solid that can be redisolved insterile water for injection. Examples of suitable protocols of conjugateadministration are as follows. Conjugates are given weekly for 6 weeksas an i.v. bolus. Bolus doses are given in 50 to 400 ml of normal salineto which human serum albumin (e.g. 0.5 to 1 mL of a concentratedsolution of human serum albumin, 100 mg/mL) can be added. Dosages willbe about 50 μg to 10 mg/kg of body weight per week, i.v. (range of 10 μgto 100 mg/kg per injection). Six weeks after treatment, the patient mayreceive a second course of treatment. Specific clinical protocols withregard to route of administration, excipients, diluents, dosages, times,etc., can be determined by the skilled artisan as the clinical situationwarrants.

[0092] Examples of medical conditions that can be treated according tothe in vivo or ex vivo methods of killing selected cell populationsinclude malignancy of any type including, for example, cancer of thelung, breast, colon, prostate, kidney, pancreas, ovary, and lymphaticorgans; melanomas; autoimmune diseases, such as systemic lupus,rheumatoid arthritis, and multiple sclerosis; graft rejections, such asrenal transplant rejection, liver transplant rejection, lung transplantrejection, cardiac transplant rejection, and bone marrow transplantrejection; graft versus host disease; viral infections, such as CMVinfection, HIV infection, AIDS, etc.; bacterial infection; and parasiteinfections, such as giardiasis, amoebiasis, schistosomiasis, and othersas determined by one skilled in the art.

EXAMPLES

[0093] The invention will now be illustrated by reference tonon-limiting examples. Unless otherwise stated, all percents, ratios,parts, etc. are by weight.

[0094] Materials and Methods

[0095] Melting points were measured using an Electrothermal apparatusand are uncorrected. NMR spectra were recorded on a Bruker AVANCE400(400 MHz) spectrometer. Chemical shifts are reported in ppm relative toTMS as an internal standard. Mass spectra were obtained using a BrukerEsquire 3000 system. Ultraviolet spectra were recorded on a HitachiU1200 spectrophotometer. HPLC was performed using a Beckman Coulter GOLD125 system equipped with a Beckman Coulter system GOLD 168 variablewavelength detector and a Waters RADIALPAK, (a reverse phase C-18column). Thin layer chromatography was performed on Analtech GF silicagel TLC plates. Silica gel for flash column chromatography was fromBaker. Tetrahydrofuran was dried by distillation over sodium metal.Dimethylactamide and dimethylformamide were dried by distillation overcalcium hydride under reduced pressure. All other solvents used werereagent grade or HPLC grade.

[0096] The synthesis of prodrugs DC2 (2), DC3 (3) and DC4 (4) DC5 DC6DC7 DC8 (FIGS. 2-6) is described herein. DC2, DC3 and DC4 are derivedfrom the parent drug DC1, while DC6, DC7 and DC8 can be prepared fromthe pegylated parent drug DC5. The prodrugs DC2 and DC3 are extremelystable in aqueous solutions, and can be converted into the parent drugDC1 by incubation in serum, plasma or with an enzyme such as carboxylesterase. These drugs also have enhanced water solubility as comparedwith DC1. The prodrug DC4 is also extremely stable in aqueous solutionsand also soluble. Incubation of DC4 with alkaline phosphatase convertsit into the parent drug DC1.

[0097] The synthetic scheme for the conversion of DC1 (1) to theprodrugs DC2 (2) and DC3 (3) is shown in FIG. 4. The phenolicsubstituent on DC1 can be reacted with any one of the reagents listed inFIG. 3 to give the intermediate 5. Reaction of 5 with N-methylpiperazineprovides DC2. Reaction of 5 with 4-piperidino-piperidine provides DC3.

[0098] DC1 was converted to the prodrug DC4 as shown in FIG. 6.Treatment of DC1-SMe with dibenzylphosphate and carbon tetrachloride inthe presence of base provided the intermediate 4 c, while reaction ofDC1-SMe with phosphorous oxychloride provide intermediate 4 b. Removalof the benzyl protecting groups of 4 c with hydrogen, with concomitantreduction of the disulfide bond provided DC4. Reduction of intermediate4 b with TCEP or DTT provided DC4.

Example I Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-methylpiperazino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC2-SMe, 2 b)

[0099] To a solution of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC1SMe) DC1-SMe (1 b, 40 mg, 0.058 mmol) in THF (4 mL) was added4-nitrophenyl chloroformate (17 mg, 0.084 mmol) anddi-isopropylethylamine (DIPEA, 15 μl). The reaction mixture was stirredunder an Argon atmosphere for 3 h. Analysis by TLC showed all DC1 hadbeen consumed to form an intermediate with an Rf value of 0.45 (mobilephase of 1:2 Acetone/Toluene). The reaction mixture was treated with4-methylpiperazine (8.3 mg, 0.084 mmol), and then stirred overnightunder Argon. The mixture was then diluted with a 1:1 (v/v)} mixture ofEtOAc/THF (15 mL) and aqueous 1 M NaH₂PO₄, pH 5.0 (5 mL). The organiclayer was separated, and the aqueous layer was extracted with EtOAc/THF(1:1, 4 ×15 ml). The organic layers were combined, dried over MgSO₄,filtered, evaporated, purified by silica gel chromatography, elutingwith acetone/toluene, (3:8) and recrystallized with THF/EtOAc/Hexane toafford 40 mg (85% yield) of DC2-SMe (2b). Rf=0.31 (Acetone/Toluene,3:8); mp=225° C. (dec.); ¹H NMR (DMF-d7) 11.78 (s, 1H), 11.70 (s, 1H),10.27 (s, 1H), 10.03 (s, 1H), 8.42 (d, 1H, J=1.7 Hz), 8.38 (s, 1H), 8.22(d, 1H, J=1.7 Hz), 8.12 (d, 1H, J=8.4 Hz), 7.97 (d, 1H, J=8.2 Hz), 7.72(dd, 1H, J=1.9, 8.8 Hz), 7.65 (dt, 1H, J=1.1, 7.0 +7.0 Hz), 7.60 (d, 1H,J=8.9 Hz), 7.54 (t, 2H, J=7.4+8.6 Hz), 7.48 (d, 1H, J=1.4 Hz), 7.44 (dd,1H, J=1.9, 8.8 Hz), 7.36 (d, 1H, J=1.6 Hz), 5.01 (t, 1H, J=10.0 Hz),4.84 (dd, 1H, J=2.2, 10.9 Hz), 4.49 (m, 2H), 4.21 (dd, 1H, J=3.2,11.3Hz), 4.10 (m, 2H), 3.82 (m, 2H), 3.22 (m, 2H), 3.13 (t, 2H,J=7.0Hz), 2.96 (m, 2H), 2.87 (t, 2H, J=7.1 Hz), 2.50 (s, 3H), 2.31 (s,3H); ¹³C NMR 169.52, 161.13, 160.41, 153.73, 148.70, 142.72, 134.67,134.49, 133.67, 133.36, 133.15, 132.16, 130.64, 128.41, 128.33, 128.22,125.66, 125.59, 124.06, 123.10, 122.92, 119.97, 118.31, 113.49, 112.93,112.90, 112.11, 111.66, 108.04, 106.98, 106.76, 103.80, 67.90, 67.65,61.79, 55.82, 48.15, 42.68, 37.01, 34.56, 34.32, 23.36; MS m/z+831.14(M+Na)⁺, 833.13, 832.15, 847.14 (M+K)⁺, 849.14, 848.14; MS m/z−807.30(M−H)−, 808.25, 809.26, 810.23.

Example II Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-methylpiperazino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC2, 2 a)

[0100] A solution of tris-(2-carboxyethyl) phosphine hydrochloride(TCEP, 30 mg, 0.104 mmol) in H₂O (2 mL) was adjusted to pH 7.0 withNaHCO₃ powder. To the solution was added 25 mg (0.031 mmol) of DC2-SMe(2 b) in DMA (3 mL). After stirring for 2 h, the pH was adjusted tobetween 3-4 by the addition of a few drops of HOAc. The mixture wasconcentrated and purified using preparative TLC on silica gel, elutingwith acetone/toluene, 1:2) to yield 21 mg (90%) of DC2 (2a). ¹H NMR(CD₃COCD₃) 10.91 (br, 2H), 10.81 (br, 1H), 9.56 (s, 1H), 9.18 (s, 1H),8.37 (s, 1H), 8.38 (s, 1H), 8.15 (m, 1H), 8.02 (d, 1H, J=8.4 Hz), 7.94(d, 1H, J=8.6 Hz), 7.63-7.55 (m, 3H), 7.47 (m, 1H), 7.36 (dd, 1H, J=2.1,8.8 Hz), 7.29 (m, 1H), 7.21 (m, 1H), 5.44 (dd, 1H, J=2.0, 5.9 Hz), 4.85(m, 2H), 4.40 (m, 2H), 4.10 (dd, 1H, J=3.2, 11.2 Hz), 3.92 (dd, 1H,J=7.8, 11.3 Hz), 3.81 (m, 2H), 3.22 (m, 2H), 3.08 (t, 2H, J=7.0 Hz),2.87 (m, 2H), 2.82 (t, 2H, J=7.1 Hz), 2.38 (s, 3H); MS m/z+785.22(M+Na), 786.20, 787.20, 801.14 (M+K), 803.16 (M+2+K); MS m/z−762.10(M−H), 764.05, 763.08.

Example III Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-piperidino-piperidino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methydithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC3-SM2, 3 b). (3 b) (DC3-SMe).

[0101] To a solution of DC1-SMe, 1 b, (50 mg, 0.073 mmol) in THF (4 ml)was added 4-nitrophenyl chloroformate (35 mg, 0.173 mmol) and DIPEA (50μl). After stirring under Argon for 3 h , TLC analysis showed that allthe DC1-SMe had been consumed to give an intermediate with Rf=0.45, 1:2Acetone/Toluene). The reaction mixture was treated with4-piperidino-piperidine (40 mg, 0.21 mmol), resulting in the formationof a heavy precipitate. The mixture was allowed to stir for 4 h, dilutedwith 20 ml of EtOAc/THF (1:1) and 5 ml of 1 M NaH₂PO₄, pH 4.5. Theorganic layer was separated, and the aqueous layer was extracted withEtOAc/THF (1:1, 4×15 ml). The organic layers were combined, dried overMgSO₄, filtered, evaporated, purified with Silica gel chromatography(Acetone/Toluene, 3:8) and crystallized with THF/EtOAc/Hexane to affordDC3-SMe (3 b, 45 mg, 70% yield), mp=285° C. (dec.); [α]=29.7° (c 0.5 inDMF); ¹H NMR 11.93 (s, 1H), 11.76 (s, 1H), 10.44 (s, 1H), 10.09 (s, 1H),8.45 (s, 1H), 8.36 (s, 1H), 8.22 (s, 1H), 8.11 (d, 1H, J=8.3 Hz), 8.00(d, 1H, J=8.4 Hz), 7.79 (dd, 1H, J=1.4, 8.6 Hz), 7.65 (t, 1H, J=7.5 Hz),7.61-7.52 (m, 3H), 7.47-7.44 (m, 2H), 7.35 (d, 1H, J=1.0 Hz), 5.00 (t,1H, J=10.0 Hz), 4.83 (dd, 1H, J=1.2, 10.2 Hz), 4.67 (m, 1H), 4.51 (m,1H), 4.30 (m, 1H), 4.21 (dd, 1H, J=3.1, 11.1 Hz), 4.11 (m, 1H), 3.40 (m,2H), 3.12 (t, 2H, J=7.0 Hz), 3.10 (m, 2H), 2.95 (m, 1H), 2.87 (t, 2H,J=7.1 Hz), 2.49 (s, 3H), 2.39 (m, 2H), 2.12 (m, 2H), 2.02-1.60 (m, 10H);¹³C NMR 169.52, 162.90, 161.15, 160.34, 153.55, 148.47, 142.66, 134.66,134.48, 133.76, 133.35, 133.29, 132.08, 130.63, 128.37, 128.22, 125.69,125.52, 123.47, 123.11, 120.01, 118.01, 113.43, 112.89, 112.08, 111.72,63.44, 60.08, 56.01, 50.00, 48.08, 42.62, 37.01, 34.07, 24.10, 24.08,23.08, 22.78; MS m/z+878.24 (M+H)⁺, 880.24, 879.25, 880.24; MSm/z−876.40 (M−H)⁻, 878.34, 877.37, 879.35.

Example IV Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-piperidino-piperidino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC3, 3 a). (3 a) (DC3).

[0102] A solution of TCEP (15.2 mg, 0.053 mmol) in H₂O (0.7 mL) wasadjusted to pH 7.0 by the addition of 13.5 mg of NaHCO₃ powder. To thesolution was added DC3-SMe (3 b, 8.2 mg, 0.0093 mmol) in DMA (2 mL), andthe reaction mixture was stirred for 2 h. A few drops of HOAc was thenadded to adjust the pH to between 3-4. The mixture was concentrated andpurified using preparative TLC on silica gel, eluting withacetone/toluene, (1:2) to yield 7 mg of DC3 (3 a). MS m/z+854.31(M+Na)⁺, 855.31, 856.32, 857.31.

Example V Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(phosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(4 b) (DC4-SMe)

[0103] A solution of DC1-SMe (1 b, 50 mg, 0.073 mmol) in a mixture ofTHF (5 ml), CH₃CN (4 ml) and DMA (0.5 ml) was stirred under anatmosphere of Argon. To the mixture were sequentially added POCl₃ (80μL), DIPEA (150 μL) and DMAP (3 mg). After stirring for 2 h, both TLCand HPLC analyses showed that the DC1-SMe had been completely consumed.Aqueous 1.0 M NaH₂PO₄, pH 4.0 (2 ml) was added, and the mixture wasstirred overnight. The mixture was further acidified with H₃PO₄ to pH2.0, saturated with NaCl, and extracted with THF/EtOAc (1:1, 6×15 ml).The organic layer was separated, concentrated and the residue wasrecrystallized with THF/H₂O/CH₃OH to afford 47 mg (84%) of the titlecompound (DC4-SMe). ¹ NMR (DMF-d7) 11.77 (s, 1H), 11.70 (s, 1H), 10.26(s, 1H), 10.02 (s, 1H), 8.74 (s, 1H), 8.42 (s, 1H), 8.30 (d, 1H, J=7.6Hz), 8.22 (s, 1H), 7.72 (d, 1H, J=8.3 Hz), 7.59 (m, 2H), 7.55-7.43 (m,4H), 7.33 (s, 1H), 4.96 (t, 1H, J=9.8 Hz), 4.81 (d, 1H, J=10.2 Hz), 4.42(m, 1H), 4.18 (m, 1H), 4.05 (dd, 1H, J=7.8, 11.0Hz), 3.11 (t, 2H,J=7.0Hz), 2.87 (t, 2H, J=7.1 Hz), 2.49 (s, 3H); ¹³C NMR 169.96, 161.45,160.85, 143.10, 135.12, 134.93, 134.15, 133.81, 133.58, 132.80, 131.60,128.86, 128.78, 128.02, 124.50, 124.05, 120.22, 118.61, 113.97, 113.40,113.32, 112.57, 108.02, 104.27, 56.18, 48.60, 43.21, 37.47, 34.49,23.53; ³¹P NMR −3.37; MS m/z 762.77 (M−H)⁻, 764.80, 763.76.

Example VI Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(phosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide(4 a) (DC4)

[0104] A solution of TCEP (30 mg, 0.104 mmol) in 2 ml of H₂O wasadjusted to pH 6.5-7.0 by the addition of NaHCO₃ powder. To the solutionwas added DC4-SMe (26 mg, 0.034 mmol) in 3 ml of DMA/ H₂O (1:1). Afterbeing stirred for 2 h under Argon, a few drops of 10% H₃PO₄ was added toreach pH 2.0. The mixture was then extracted with DMA/EtOAc (1:5, 6×10ml). The organic layers were combined, evaporated and purified bypreparative HPLC, using a C18 column, 20×250 mm, flow rate=8.0 ml/min.,mobile phase: A: 0.01% HOAc in H₂O, B: 2% DMA in CH₃CN; time table:0-10′, 5% B; to 20′, 20% B; to 50′:50% B. The DC4 peak eluted between30-38 min.. The fractions containing DC4 were pooled, concentrated anddried under vacuum to yield 22 mg (89%) of the title compound 4a. ¹H NMR(DMF-d7) 11.76 (s, 1H), 11.69 (s, 1H), 10.26 (s, 1H), 10.02 (s, 1H),8.78 (s, 1H), 8.41 (s, 1H), 8.29 (d, 1H, J=8.3 Hz), 8.21 (s, 1H), 7.79(d, 1H, J=5.1 Hz), 7.60-7.43 (m, 6H), 7.27 (s, 1H), 4.96 (t, 1H, J=9.2Hz), 4.80 (d, 1H, J=10.6 Hz), 4.42 (m, 1H), 4.23 (dd, 1H, J=2.2,9.4 Hz),4.06 (dd, 1H, J=7.4, 10.6 Hz), 3.12 (t, 2H, J=7.1 Hz), 2.88 (t, 2H,J=7.1 Hz); MS m/z 716.16 (M−H)⁻, 717.12, 718.12.

[0105] Alternatively, to a solution of DTT (20 mg) in a mixture ofacetone (3 mL) and 50 mM NaH₂PO₄ buffer (3 mL), pH 7.0 was added DC4SMe(22 mg, 0.028 mmol). After stirring under Argon for 4 h, a few drops of5% H₃PO₄ was added to pH 3.0. The mixture was concentrated and purifiedon a C18 column (1.0×12 cm) eluted with 100% water to 50% water inacetone. The fractions were pooled and evaporated to dryness to afford18 mg (90%) of DC4 (4 a). MS m/z−716.30, 718.30, 717.30.

Example VII Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(dibenzylphosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide(4 c) (DC4-SMe Dibenzylphosphate)

[0106] To a solution of DC1-SMe (50 mg, 0,073 mmol) in 10 ml ofTHF/CH₃CN (1:1) under argon were sequentially added CCl₄ (100 μL, 1.036mmol), DIPEA (55 μL, 0.316 mmol), dibenzylphosphite (100 μL, 0.452 mmol)and DMAP (0.2 mg, 0.0016 mmol). After stirring overnight under Argon,the reaction appeared complete by TLC analysis, with a new product beingformed with an Rf=0.37 in acetone/toluene 1:2). The mixture was dilutedwith 5 ml of 1.0 M NaH₂PO₄,pH 4.0, and EtOAc (10 mL). The organic layerwas separated and the aqueous solution was extracted with THF/EtOAc(1:1, 4×15 ml). The organic layers were combined, dried over MgSO₄,filtered, evaporated and purified by silica gel chromatography, elutingwith acetone/toluene (3:7), to afford 62 mg (89%) of the title compound4 c. ¹H NMR (DMF-d7) 11.84 (s, 1H), 11.74 (s, 1H), 10.31 (s, 1H), 10.04(s, 1H), 8.77 (s, 1H), 8.44 (s, 1H), 8.22 (s, 1H), 8.10 (t, 2H, J=7.3Hz), 7.74 (dd, 1H, J=1.7, 8.8 Hz), 7.66-7.61 (m, 2H), 7.55-7.29 (m,15H), 5.37 (t, 4H, J=7.5 Hz), 5.01 (m, 2H), 4.84 (dd, 1H, J=1.9, 10.9Hz), 4.51 (m, 1H), 4.20 (dd, 1H, J=3.2, 10.9Hz), 4.11 (dd, 1H, J=6.9,11.1 Hz), 3.13 (t, 2H, J=7.2Hz), 2.87 (t, 2H, J=7.1 Hz), 2.50 (s, 3H);¹³C NMR 169.52, 161.18, 160.41, 147.88, ,147.20, 136.74, 136.67, 134.68,134.56, 133.70, 133.36, 133.20, 132.11, 130.83, 129.25, 129.23, 129.21,129.19, 128.84, 128.78, 128.62, 128.42, 128.32, 127.90, 124.28, 124.22,123.96, 123.33, 122.87, 120.03, 118.32, 113.51, 112.94, 112.12, 108.42,106.87, 70.75, 70.69,, 67.91, 55.90, 47.96, 42.59, 37.02, 34.04, 23.08;³¹P NMR −4.49; MS m/z 966.17 (M+Na)⁺, 968.14 (M+Na)⁺, 967.17.

[0107] Conversion to DC4:

[0108] A flask was charged with 4 c (20 mg, 0.021 mmol), and treatedwith Pd/C (15 mg), glacial acetic acid (100 μl) and DMA (4 ml). Thesystem was evacuated with vacuum suction, and then stirred underhydrogen through a hydrogen-filled balloon overnight. The catalyst wasremoved by filtration and the solvent was evaporated, and the residuewas purified by preparative HPLC as above described to yield 6 mg (39%)of DC4 (4 a). MS m/z 716.48 (M−H)⁻, 717.48, 718.50.

Example VIII Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-nitro-1H-indole-2-carboxamide(DC0, 10 a)

[0109] To a solution of5-hydroxy-3-amino-1-[S]-(chloromethyl)-1,2-dihydro-3H-benz(e)indole,hydrochloride salt [seco-(−) CBI, 20 mg, 0.72 mmol] and5-[5′-nitroindol-2′-yl-carbonyl amino]indole-2-carboxylic acid (9, 25mg, 0.068 mmol) in DMA (3 mL) was added EDC (40 mg, 0.20 mmol) underArgon. After stirring overnight, a few drops of 50% HOAc were added andthe mixture was evaporated to dryness and purified by preparative silicagel TLC chromatography (40% acetone in toluene) to afford 25 mg of DC0(10 a). ¹H NMR (DMF-d₇) 12.54 (s, 1H), 11.73 (s, 1H), 10.60 (s, 1H),10.58 (s, 1H), 8.80 (d, 1H, J=2.3 Hz), 8.42 (d, 1H, J=1.9 Hz), 8.25 (d,1H, J=8.5 Hz), 8.19 (dd, 1H, J=2.1, 9.1 Hz), 8.09 (br, 1H), 7.95 (d, 1H,J=8.3 Hz), 7.82 (d, 1H, J=1.5 Hz), 7.79 (d, 1H, J=9.1 Hz), 7.74 (dd, 1H,J=2.0, 8.9 Hz), 7.62 (d, 1H, J=8.8 Hz), 7.58 (dt, 1H, J=1.7, 7.0+7.0Hz), 7.42 (dt, 1H, J=1.2, 7.0+7.0 Hz), 7.33 (d, 1H, J=1.7 Hz), 4.91 (t,1H, J=11.0 Hz), 4.77 (dd, 1H, J=2.1, 11.1 Hz), 4.33 (m, 1H), 4.13 (dd,1H, J=3.1, 11.1 Hz), 3.97 (dd, 1H, J=7.9, 11.1 Hz); ¹³C NMR 163.35,161.48, 160.05, 155.79, 142.98, 137.18, 135.03, 133.22, 133.16, 131.50,128.85, 128.45, 128.11, 124.62, 124.02, 123.76, 120.33, 119.36, 118.70,116.45, 114.00, 113.08, 106.97, 105.02, 101.53; MS m/z 602.96 (M+Na)⁺,604.78, 603.81, 618.64 (M+K)⁺, 620.48.

Example IX Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide,DC1SMe (1 b)

[0110] A flask was charged with 10 a (10 mg, 0.017 mmol), Pd/C (10 mg),HCl (conc. 3 μl) and DMA (2.5 ml). After the air was evacuated, hydrogenwas conducted by hydrogen balloon overnight. The catalyst was removed byfiltration and the solvent was evaporated to give 10 b as a brown solid.The solid compound was used directly without further purification.

[0111] To 10 b in DMA (2 mL) was added 3-(methyldithio)propionic acid (5mg, 0.032 mmol) and EDC (15 mg, 0.078 mmol) under Argon. After stirringovernight, two drops of 50% HOAc were added, and the mixture wasevaporated to dryness and purified by preparative silica gel TLC (40%acetone in toluene) to afford 6 mg of DC1-SMe (1 b) R_(f)=0.40 (3:7Acetone/Toluene); ¹H NMR (CD₃COCD₃) 10.91 (s, 1H), 10.88 (s, 1H), 9.64(s, 1H), 9.56 (s, 1H), 9.27 (s, 1H), 8.35 (d, 1H, J=1.9 Hz), 8.25 (d,1H, J=8.0 Hz), 8.17 (d, 1H, J=1.9 Hz), 8.07 (s, 1H), 7.88 (d, 1H, J=8.3Hz), 7.64 (dd, 1H, J=2.0, 8.1 Hz), 7.58-7.50 (m, 3H), 7.38-7.35 (m, 2H),7.31 (d, 1H, J=1.7 Hz), 7.26 (d, 1H, J=1.7 Hz), 4.86 (dd, 1H, J=8.7,11.0 Hz), 4.80 (dd, 1H, J=2.3, 10.9 Hz), 4.30 (m, 1H), 4.07 (dd, 1H,J=3.1, 11.0 Hz), 3.83 (dd, 1H, J=8.4, 11.2 Hz), 3.09 (t, 2H, J=7.1 Hz),2.83 (t, 2H, J=7.1 Hz), 2.45 (s, 3H); ¹³C NMR 169.56, 161.10, 160.43,155.13, 143.50, 134.78, 134.46, 133.55, 133.34, 133.03, 132.57, 131.21,128.80, 128.69, 128.21, 124.22, 124.02, 123.53, 123.44, 120.16, 118.79,116.45, 113.91113.02, 112.95, 112.73, 106.78, 103.72, 101.63, 56.01,47.73, 43.10, 37.25, 34.01, 23.00; MS m/z 706.71 (M+Na)⁺, 708.58,707.71, 722.34 (M+K)⁺, 724.42.

Example X Preparation of(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(15″-methyldithio-4″,7″,10″,13″-tetraoxapentadecyl-1-oxopropyl)-amino]-1H-indole-2-carboxamide(DC5-SMe)

[0112] To a solution of 10 b (50 mg, 0.091 mmol) in DMA (5 mL) was added15″-methyldithio-4″,7″,10″,13″-tetraoxapentadecanoic acid (33 mg 0.100mmol) and EDC (88 mg, 0.459 mmol) under Ar. After being stirredovernight, two drops of 50% HOAc were added to the mixture and themixture was evaporated to dryness, purified by Silica gel chromatography(30% acetone in toluene) to afford DC5-SMe

[0113] Certain patents and printed publications have been referred to inthe present disclosure, the teachings of which are hereby eachincorporated in their respective entireties by reference.

[0114] While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madethereto without departing from the spirit and scope thereof.

What is claimed is:
 1. A prodrug comprising an analog of CC-1065 inwhich the phenolic group of the alkylating portion of the molecule isprotected and wherein said prodrug further comprises a linker capable ofconjugating said prodrug to a cell binding agent.
 2. The prodrug ofclaim 1 wherein said linker comprises a thiol or a disulfide bond. 3.The prodrug of claim 1 wherein said protecting group increaseswater-solubility of said prodrug.
 4. The prodrug of claim 3 wherein saidprotecting group is selected from the group consisting of a piperazinocarbamate, a 4-piperidino-piperidino carbamate and a phosphate.
 5. Theprodrug of claim 1 wherein said linker comprises a polyethylene glycolof the formula —(—O(CH2)2)_(n)—, wherein n is an integer from 2 to 1000.6. A composition comprising the prodrug of claim 1 and apharmaceutically acceptable carrier.
 7. A prodrug comprising an analogof a seco-cyclopropabenzindole-containing cytotoxic drug selected fromthe group consisting of analogs formed from a first subunit of formula(I) covalently linked to a second subunit of the formula (II), (III),(IV), (V), (VI), (VII), (VIII) or (IX) via an amide bond from thesecondary amino group of the pyrrole moiety of the first subunit to theC-2 carboxyl of the second subunit, wherein the formula (I) is asfollows:

wherein the formulae (II)-(IX) are as follows:

wherein R represents a moiety that enables linkage of said prodrug to acell binding agent; wherein R₁-R₆ are each independently hydrogen, C₁-C₃linear alkyl, methoxy, hydroxyl, primary amino, secondary amino,tertiary amino, or amido; and wherein R₇ is an enzyme-cleavableprotecting group.
 8. The prodrug of claim 7, wherein R comprises a thiolor a disulfide bond.
 9. The prodrug of claim 7, wherein R₁-R₆ arehydrogen.
 10. The prodrug of claim 7, wherein R₇ is selected from thegroup consisting of a piperazino carbamate, a 4-piperidino-piperidinocarbamate and a phosphate.
 11. The prodrug of claim 10, wherein Rrepresents a moiety that enables linkage of the prodrug to a cellbinding agent via a disulfide bond.
 12. The prodrug of claim 10, whereinsaid second subunit is represented by formula (II) and wherein R₁-R₆ arehydrogen.
 13. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-methylpiperazino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 14. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-methylpiperazino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 15. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-piperidino-piperidino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methydithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 16. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-[(4-piperidino-piperidino)carbonyloxy]-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 17. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(phosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 18. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(phosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-mercapto-1-oxopropyl)-amino]-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 19. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-(dibenzylphosphonoxy)-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 20. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-nitro-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 21. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(3-methyldithio-1-oxopropyl)-amino]-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 22. The prodrug of claim 12 which is(S)-N-[2-{(1-chloromethyl)-1,2-dihydro-5-hydroxy-3H-benz(e)indol-3-yl}carbonyl]-1H-indol-5-yl]-5-[(15″-methyldithio-4″,7″,10″,13″-tetraoxapentadecyl-1-oxopropyl)-amino]-1H-indole-2-carboxamide,or a salt or an isomer thereof.
 23. A prodrug conjugate comprising acell binding agent linked to one or more of the prodrugs of claim 1 orclaim
 7. 24. The prodrug conjugate of claim 23 wherein said cell bindingagent is an antibody or a fragment thereof.
 25. A composition comprisingthe prodrug of claim 7 and a pharmaceutically acceptable carrier.
 26. Amethod for treating a subject, comprising administering to a subject inneed thereof an effective amount of the composition of claim 6 or 25.27. A method for treating a subject, comprising administering to asubject in need thereof an effective amount of the prodrug conjugate ofclaim
 24. 28. The prodrug of claim 7 wherein said linker comprisespolyethylene glycol of the formula —(—O(CH₂)₂)_(n)—, wherein n is aninteger from 2 to 1000.